Method of Analyzing the Accuracy of a Sequence of Probe Nucleic Acid Immobilized on a Microarray Substrate

Abstract

A method of analyzing an accuracy of a sequence of a probe nucleic acid immobilized in a microarray includes providing a microarray in which regions where a probe nucleic acid is immobilized on a substrate are arrayed, hybridizing the probe nucleic acid with a target nucleic acid that is complementary to the probe nucleic acid to form a hybridization product, wherein the target nucleic acid is labeled with a detectable signal material on at least one end, reacting the hybridization product with an enzyme to remove the detectable signal material from the target nucleic acid, wherein the at least one end of the target nucleic acid remains unpaired with the probe nucleic acid, measuring a residual signal generated from the resultant enzyme reaction product, comparing the measured signal value with a signal value generated from a control group experiment; and analyzing an accuracy of the probe nucleic acid sequence.

1 . A method of analyzing an accuracy of a sequence of a probe nucleic acid immobilized in a microarray, the method comprising:
 providing a microarray in which regions where a probe nucleic acid is immobilized on a substrate through a 3′ end or a 5′ end are arrayed;   hybridizing the probe nucleic acid with a target nucleic acid that is complementary to the probe nucleic acid to form a hybridization product, wherein the target nucleic acid is labeled with a detectable signal material at at least one end selected from a group comprising a 3′ end and a 5′ end;   reacting the hybridization product with at least one enzyme selected from 3′ exonuclease and 5′ exonuclease to remove the detectable signal material from the at least one end of the target nucleic acid, wherein the at least one end of the target nucleic acid remains unpaired with the probe nucleic acid;   measuring a residual signal generated from the resultant enzyme reaction product;   comparing the measured signal value with a signal value generated from a control group experiment; and   analyzing an accuracy of the probe nucleic acid sequence.   
     
     
         2 . The method of  claim 1 , wherein the probe nucleic acid is immobilized on the substrate through the 3′ end, and the 5′ end of the probe nucleic acid is exposed. 
     
     
         3 . The method of  claim 1 , wherein the probe nucleic acid is immobilized on the substrate through the 5′ end, and the 3′ end of the probe nucleic acid is exposed. 
     
     
         4 . The method of  claim 1 , wherein the signal material is selected from a fluorescent material or a radioactive material. 
     
     
         5 . The method of  claim 1 , wherein the detectable signal material comprises a signal generating material and a quencher, wherein
 at least one end selected from 3′ and 5′ ends of the target nucleic acid is labeled with the signal generating material, and the other end of the target nucleic acid is labeled with the quencher, wherein,   when the quencher is removed by the exonuclease, a signal is generated.   
     
     
         6 . The method of  claim 4 , wherein the fluorescent material is selected from Cy3 and Cy5. 
     
     
         7 . The method of  claim 1 , wherein the length of the target nucleic acid is the same as an intended length of the probe nucleic acid. 
     
     
         8 . The method of  claim 1 , wherein the probe nucleic acid is synthesized on the substrate by photolithography. 
     
     
         9 . The method of  claim 1 , wherein the exonuclease is a single strand specific exonuclease. 
     
     
         10 . The method of  claim 1 , wherein the exonuclease is selected from a group comprising a DNA polymerase I, a Klenow fragment and a T4 DNA polymerase. 
     
     
         11 . The method of  claim 1 , wherein the control group experiment is performed using the method of  claim 1 , except that the reacting step is not used 
     
     
         12 . The method of  claim 1 , wherein the control group experiment is performed using the method of  claim 1 , except for use of a microarray in which a probe nucleic acid that has been synthesized in a liquid phase is immobilized by spotting. 
     
     
         13 . The method of  claim 1 , wherein, when the measured signal value is equal to or less than a predetermined level of a signal value obtained from the control group experiment, the comparing step further comprises determining that the probe nucleic acid of the microarray is defective. 
     
     
         14 . The method of  claim 1 , wherein, when the measured signal value is equal to or greater than a predetermined level of a signal value obtained from the control group experiment, the comparing step further comprises determining that the probe nucleic acid of the microarray is defective 
     
     
         15 . A method of analyzing an accuracy of a sequence of a probe nucleic acid immobilized in a microarray, the method comprising:
 providing a microarray in which regions where a probe nucleic acid is immobilized on a substrate through a 3′ end or a 5′ end are arrayed;   hybridizing the probe nucleic acid with a target nucleic acid that is complementary to the probe nucleic acid to form a hybridization product, wherein the target nucleic acid is labeled with a detectable signal material, wherein the detectable signal material comprises a signal generating material and a quencher, wherein at least one end selected from 3′ and 5′ ends of the target nucleic acid is labeled with the signal generating material, and the other end of the target nucleic acid is labeled with the quencher;   reacting the hybridization product with at least one enzyme selected from 3′ exonuclease and 5′ exonuclease to remove the detectable signal material from the target nucleic acid, wherein at least one end of the target nucleic acid remains unpaired with the probe nucleic acid, and wherein, when the quencher is removed by the exonuclease, a residual signal is generated   measuring said residual signal generated from the resultant enzyme reaction product;   comparing the measured residual signal value with a signal value generated from a control group experiment; and   analyzing an accuracy of the probe nucleic acid sequence.   
     
     
         16 . The method of  claim 15 , wherein the target nucleic acid is labeled with said detectable signal material at at least one end selected from a group comprising a 3′ end and a 5′ end, and the detectable signal material is removed from the at least one end of the target nucleic acid. 
     
     
         17 . A method of analyzing an accuracy of a sequence of a probe nucleic acid immobilized in a microarray, the method comprising:
 providing a microarray in which regions where a probe nucleic acid is immobilized on a substrate through a 3′ end or a 5′ end are arrayed;   hybridizing the probe nucleic acid with a target nucleic acid that is complementary to the probe nucleic acid to form a hybridization product, wherein a length of the target nucleic acid is the same as an intended length of the probe nucleic acid, and the target nucleic acid is labeled with a detectable signal material at at least one end selected from a group comprising a 3′ end and a 5′ end;   reacting the hybridization product with at least one enzyme to remove the detectable signal material from the at least one end of the target nucleic acid, wherein the at least one end of the target nucleic acid remains unpaired with the probe nucleic acid;   measuring a residual signal generated from the resultant enzyme reaction product;   comparing the measured signal value with a signal value generated from a control group experiment; and   analyzing an accuracy of the probe nucleic acid sequence.   
     
     
         18 . The method of  claim 17 , wherein the at least one enzyme is selected from 3′ exonuclease and 5′ exonuclease.